ATP-competitive mTOR Inhibitor Research Articles

Identification of Brain-Penetrant ATP-Competitive mTOR Inhibitors for CNS Syndromes.

The allosteric inhibitor of the mechanistic target of rapamycin (mTOR) everolimus reduces seizures in tuberous sclerosis complex (TSC) patients through partial inhibition of mTOR functions. Due to its limited brain permeability, we sought to develop a catalytic mTOR inhibitor optimized for central nervous system (CNS) indications. We recently reported an mTOR inhibitor (1) that is able to block mTOR functions in the mouse brain and extend the survival of mice with neuronal-specific ablation of the Tsc1 gene. However, 1 showed the risk of genotoxicity in vitro. Through structure-activity relationship (SAR) optimization, we identified compounds 9 and 11 without genotoxicity risk. In neuronal cell-based models of mTOR hyperactivity, both corrected aberrant mTOR activity and significantly improved the survival rate of mice in the Tsc1 gene knockout model. Unfortunately, 9 and 11 showed limited oral exposures in higher species and dose-limiting toxicities in cynomolgus macaque, respectively. However, they remain optimal tools to explore mTOR hyperactivity in CNS disease models.

Investigation of Macrocyclic mTOR Modulators of Rapamycin Binding Site via Pharmacoinformatics Approaches

The PI3K/Akt/mTOR is an essential intracellular signaling pathway in which the serine/threonine mTOR kinase portrays a major role in cell growth, proliferation and survival. The mTOR kinase is frequently dysregulated in a broad spectrum of cancers, thus making it a potential target. Rapamycin and its analogs (rapalogs) allosterically inhibit mTOR, thereby dodging the deleterious effects prompted by ATP-competitive mTOR inhibitors. However, the available mTOR allosteric site inhibitors exhibit low oral bioavailability and suboptimal solubility. Bearing in mind this narrow therapeutic window of the current allosteric mTOR inhibitors, an in silico study was designed in search of new macrocyclic inhibitors. The macrocycles from the ChemBridge database (12,677 molecules) were filtered for their drug-likeness properties and the procured compounds were subjected for molecular docking within the binding cleft between FKBP25 and FRB domains of mTOR. The docking analysis resulted with 15 macrocycles displaying higher scores than the selective mTOR allosteric site inhibitor, DL001. The docked complexes were refined by subsequent molecular dynamics simulations for a period of 100 ns. Successive binding free energy computation revealed a total of 7 macrocyclic compounds (HITS) demonstrating better binding affinity than DL001, towards mTOR. The consequent assessment of pharmacokinetic properties resulted in HITS with similar or better properties than the selective inhibitor, DL001. The HITS from this investigation could act as effective mTOR allosteric site inhibitors and serve as macrocyclic scaffolds for developing compounds targeting the dysregulated mTOR.

Abstract 4859: Bi-steric mTORC1 inhibitors are superior to rapamycin and induce apoptotic cell death in tumor models with hyperactivated mTORC1

Abstract The PI3K-mTOR pathway is one of the most commonly dysregulated pathways in human tumors. Rapalogs have been used extensively in human clinical trials but exhibit modest clinical benefit for the most part, possibly due to their lack of effect on 4E-BP1, a key target downstream of mTORC1. ATP-competitive mTOR inhibitors can fully inhibit mTORC1 but are poorly tolerated possibly due to their inhibition of mTORC2. A new class of selective mTORC1 inhibitors has been developed and termed ‘bi-steric’, which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. We report that bi-steric mTORC1-selective inhibitors (RMC-4627 and RMC-6272) have greater activity than rapalogs for tumors with mTORC1 hyperactivation. RMC-4627 and RMC-6272 showed more effective growth inhibition in multiple tumor cell lines with mTORC1 dysfunction compared to rapamycin. Both RMC-4627 and RMC-6272 at ~1 nM showed near complete inhibition of p4E-BP1T37/46, which was not seen with rapamycin treatment. Rapamycin and both bi-steric compounds markedly reduced kidney tumor burden and human bladder cancer PDX tumor size after four weeks of treatment. Tumor regrowth assessed after two-month treatment cessation was significantly reduced in the RMC-6272 group as compared to the rapamycin group. Finally, TUNEL staining showed RMC-6272 treatment led to a greater induction of apoptosis in kidney tumor cells and human bladder cancer PDX tumors relative to rapamycin after a single dose of each compound. This is the first time that clear evidence for cell death after mTORC1 inhibition has been seen. Integrative multi-omic analysis reveals differential global rewiring induced by RMC-6272 in comparison to rapamycin. Genes involved in cell cycle transition were downregulated by RMC-6272 vs. rapamycin. Proteins downregulated by RMC-6272 treatment relative to rapamycin were enriched for translation and ribosome-related proteins, consistent with effects of non-phosphorylated 4E-BP1 on synthesis of those proteins. Multiple purine metabolites were decreased to a greater extent in RMC-6272 treated cells as compared to rapamycin treatment, including deoxyadenosine, AICAR, AMP, IMP, and GMP. Notably, there was no significant change in R5P, suggesting that downregulation of the purine de novo synthesis pathway was the cause of these changes. Strong tumor cell apoptosis was induced by the suppression of de novo purine synthesis through mTORC1-PRPS1 axis. PRPS1, the rate-limit enzyme involving in purine synthesis was regulated by mTORC1 activity. In summary, RMC-4627 and RMC-6272 demonstrate improved in vitro and in vivo inhibition of mTORC1 in comparison to rapamycin, and induced more cell death in preclinical models, indicating the potential of bi-steric mTORC1-selective inhibitors as a novel therapeutic strategy to treat tumors with mTORC1 dysregulation. Citation Format: Heng Du, Yu Chi Yang, Heng-Jia Liu, Min Yuan, John Asara, Kwok-Kin Wong, Mallika Singh, David Kwiatkowski. Bi-steric mTORC1 inhibitors are superior to rapamycin and induce apoptotic cell death in tumor models with hyperactivated mTORC1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4859.

A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling

mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.

Abstract 5357: mTOR kinase inhibitor AZD8055 reverses acquired rapamycin resistance in prostate cancer cells

Abstract The mTOR pathway is frequently over-activated in human cancers. However, classic allosteric mTOR inhibitors rapamycin only exerts limited clinical benefits. It has been shown that prostate cancer are able to acquire resistance after rapamycin treatment for a period of time. Such secondary resistance remains as major concerns in rapamycin-based anti-cancer therapies that may inevitably lead to therapeutic failure. In the current study, we evaluated treatment effects of ATP-competitive dual mTOR kinase inhibitor AZD8055 on cancer cells that have acquired resistance to rapamycin, and underpinning molecular mechanisms. To establish cells with acquired rapamycin resistance, PTEN-mt prostate cancer PC-3 cells were gradually treated with increasing concentrations of rapamycin (up to 10 μM) until the cells were completely unresponsive to rapamycin (PC-3RR). Cell proliferation was determined by MTT assay and clonogenic survival was tested by colony formation assay. m7G pull-down assay was employed to detect the cap-dependent translation. mTOR downstream pathways, including p-S6K (T389)/p-S6 (S235/236), p-4EBP1 (T37/S46) and p-Akt (S473) were detected by Western Blotting. PC-3RR cells essentially failed to respond to rapamycin, with IC50 values greater than 1 μM. However, AZD8055 suppressed PC-3RR proliferation in a dose dependent manner, with IC50 < 30 nM. Similarly, clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. At the molecular level, AZD8055, but not rapamycin, strongly prevents the dissociation of 4E-BP1 from eIF4E. Further, while the p-S6K and p-S6 pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition on p-4E-BP1 and p-Akt than rapamycin. Interestingly, similar to rapamycin, AZD8055 slightly increased p-Akt (T308), suggesting the existence of the PI3K-mediated feedback loop. These findings indicate that AZD8055 significantly inhibits malignant functions in cancer cells that are highly resistant to rapamycin. Such effects are likely to be associated with the deactivation of rapamycin-insensitive mTOR downstream pathways. These data provide preliminary evidence for future evaluation of ATP-competitive mTOR inhibitors in cancer models that are resistant to rapamycin analogs. Citation Format: Yao Dai, Dietmar Siemann. mTOR kinase inhibitor AZD8055 reverses acquired rapamycin resistance in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5357.

NVP-BEZ235 Inhibits Renal Cell Carcinoma by Targeting TAK1 and PI3K/Akt/mTOR Pathways.

In spite of the promising in vitro and preclinical results, dual PI3K/Akt/mTOR inhibitor NVP-BEZ235, and ATP-competitive mTOR inhibitor PP242 both failed to confirm their inhibitory efficacy against renal cell carcinoma (RCC) in clinical settings. Therefore, a better understanding of the molecular mechanism is essential so as to provide possibilities for their use in combination with other agents. In present study, RCC cell lines (UMRC6, 786-0 and UOK121) were treated with NVP-BEZ235, PP242 or Rapamycin, an mTOR complex 1 (mTORC1)-specific inhibitor. They all suppressed cell proliferation and invasion, induced apoptosis and cell cycle arrest, and the effects were in the order of NVP-BEZ235 > PP242 > Rapamycin. Accordingly, the marked and sustained decrease in speckle-type POZ protein (SPOP) expression and phosphorylation of Akt and mTOR kinases was observed in RCC cells treated with NVP-BEZ235 and PP242, whereas only potent inhibition of mTOR activity was induced in Rapamycin-treated cells. In considering the overactivation of c-Jun and IκB-α in human renal tumor tissue, we next investigated the role of JNK and IKK pathways in the response of RCC cells to these compounds. First of all, transforming growth factor β activated kinase 1 (TAK1)-dependent activation of JNK/ (activator protein-1) AP-1 axis in RCC cells was proved by the repression of AP-1 activity with TAK1 or JNK inhibitor. Second, the profound inhibition of TAK1/JNK/AP-1 pathway was demonstrated in RCC cells treated with NVP-BEZ235 or PP242 but not Rapamycin, which is manifested as a reduction in activity of TAK1, c-Jun and AP-1. Meanwhile, subsequent to TAK1 inactivation, the activation of IκB-α was also reduced by NVP-BEZ235 and PP242. Likewise, in vivo, treatment with NVP-BEZ235 and PP242 suppressed the growth of xenografts generated from 786-0 and A498 cells, along with decreased expression of phospho-TAK1, phospho-c-Jun, and phospho-IκB-α. In contrast, Rapamycin elicited no significant inhibitory effects on tumor growth and phosphorylation of TAK1, c-Jun and IκB-α. We conclude that besides PI3K/Akt/mTOR signaling, NVP-BEZ235, and PP242 simultaneously target TAK1-dependent pathways in RCC cells. Notably, these effects were more marked in the presence of NVP-BEZ235 than PP242, indicating the potential application of NVP-BEZ235 in combination therapy for RCC.

Oxadiazol-based mTOR inhibitors with potent antiproliferative activities: synthetic and computational modeling.

Series of N-aryl-1,3,4-oxadiazole-2-amines and 3-aryl-1,2,4-oxadiazole-5-carboxamides derivatives were synthesized as novel chemotherapeutic agents. Synthesized compounds were evaluated for their anticancer activities against several cancer cell lines. Many analogues of 1,3,4-oxadiazole scaffold showed potent antiproliferative activities against breast cancer cell lines, with higher activities toward the metastatic breast cancer cell line (MDA-MB-231). Active analogues were profiled using in-house pharmacophore database in search for molecular target. Active analogues (2j and 2k) were found to fit the pharmacophoric map of ATP-competitive inhibitors of mTOR. The mTOR inhibitory activities of the most active compounds were confirmed with IC50 values in nanomolar range. The N-aryl-1,3,4-oxadiazole-2-amines linked to a basic head is a novel ATP-competitive inhibitors of mTOR with potential activities for treatment of different types of cancer.

AZD2014, a dual mTOR inhibitor, attenuates cardiac hypertrophy in vitro and in vivo

Cardiac hypertrophy is one of the most common genetic heart disorders and considered a risk factor for cardiac morbidity and mortality. The mammalian target of rapamycin (mTOR) pathway plays a key regulatory function in cardiovascular physiology and pathology in hypertrophy. AZD2014 is a small-molecule ATP competitive mTOR inhibitor working on both mTORC1 and mTORC2 complexes. Little is known about the therapeutic effects of AZD2014 in cardiac hypertrophy and its underlying mechanism. Here, AZD2014 is examined in in vitro model of phenylephrine (PE)-induced human cardiomyocyte hypertrophy and a myosin-binding protein-C (Mybpc3)-targeted knockout (KO) mouse model of cardiac hypertrophy. Our results demonstrate that cardiomyocytes treated with AZD2014 retain the normal phenotype and AZD2014 attenuates cardiac hypertrophy in the Mybpc3-KO mouse model through inhibition of dual mTORC1 and mTORC2, which in turn results in the down-regulation of the Akt/mTOR signaling pathway.

Torin 1 alleviates impairment of TFEB-mediated lysosomal biogenesis and autophagy in TGFBI (p.G623_H626del)-linked Thiel-Behnke corneal dystrophy

ABSTRACT Thiel-Behnke corneal dystrophy (TBCD) is an epithelial-stromal TGFBI dystrophy caused by mutations in the TGFBI (transforming growth factor beta induced) gene, though the underlying mechanisms and pathogenesis of TBCD are still obscure. The study identifies a novel mutation in the TGFBI gene (p.Gly623_His626del) in a TBCD pedigree. Characteristics of the typical vacuole formation, irregular corneal epithelial thickening and thinning, deposition of eosinophilic substances beneath the epithelium, and involvement of the anterior stroma were observed in this pedigree via transmission electron microscopy (TEM) and histological staining. Tgfbi-p.Gly623_Tyr626del mouse models of TBCD were subsequently generated via CRISPR/Cas9 technology, and the above characteristics were further verified via TEM and histological staining. Lysosomal dysfunction and downregulation of differential expression protein CTSD (cathepsin D) were observed using LysoTracker Green DND-26 and proteomic analysis, respectively. Hence, lysosomal dysfunction probably leads to autophagic flux obstruction in TBCD; this was supported by enhanced LC3-II and SQSTM1 levels and decreased CTSD. TFEB (transcription factor EB) was prominently decreased in TBCD corneal fibroblasts and administration of ATP-competitive MTOR inhibitor torin 1 reversed this decline, resulting in the degradation of accumulated mut-TGFBI (mutant TGFBI protein) via the ameliorative lysosomal function and autophagic flux owing to elevated TFEB activity as measured by western blot, confocal microscopy, and flow cytometry. Transfected HEK 293 cells overexpressing human full-length WT-TGFBI and mut-TGFBI were generated to further verify the results obtained in human corneal fibroblasts. Amelioration of lysosome dysfunction may therefore have therapeutic efficacy in the treatment of TBCD. Abbreviations AS-OCT: anterior segment optical coherence tomography; ATP: adenosine triphosphate; Cas9: CRISPR-associated protein 9; CLEAR: coordinated lysosomal expression and regulation; CRISPR: clustered regularly interspaced short palindromic repeats; CTSB: cathepsin B; CTSD: cathepsin D; CTSF: cathepsin F; CTSL: cathepsin L; DNA: deoxyribonucleic acid; ECM: extracellular matrix; Fas1: fasciclin 1; FC: flow cytometry; GAPDH: glyceraldeyde-3-phosphate dehydrogenase; GCD2: granular corneal dystrophy type 2; HE: hematoxylin and eosin; LAMP2: lysosomal-associated membrane protein; MT: mutation type; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; mut-TGFBI: mutant TGFBI protein; SD: standard deviation; TBCD: Thiel-Behnke corneal dystrophy; TEM: transmission electron microscopy; TFEB: transcription factor EB; TGFBI: transforming growth factor beta induced; WT: wild type

Abstract 1026: Bi-steric mTORC1-selective inhibitors demonstrate improved potency and efficacy in tumors with mTORC1 hyperactivation

Abstract The PI3K-mTOR pathway is one of the most commonly dysregulated pathways in human tumors. Rapalogs have been used extensively in human clinical trials but exhibit modest clinical benefit for the most part, possibly due to their lack of effect on 4E-BP1, a key target downstream of mTORC1. ATP-competitive mTOR inhibitors can fully inhibit mTORC1 but are poorly tolerated possibly due to their inhibition of mTORC2. A new class of selective mTORC1 inhibitors has been developed and termed ‘bi-steric', which comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. RMC-5552 is the first clinical candidate of this class and clinical testing is planned in 2021. RM-001 and RM-006 (also known as RMC-6272) are representative bi-steric tool compounds that exhibit potent and selective (> 10-fold) inhibition of mTORC1 over mTORC2. We report that bi-steric mTORC1-selective inhibitors have greater activity than rapalogs for tumors with mTORC1 hyperactivation secondary to TSC1/2 loss. RM-001 and RM-006 showed more effective growth inhibition in multiple TSC1 or TSC2 mutant tumor cell lines compared to rapamycin. RM-001 and RM-006 caused a more profound growth inhibition in the TSC1 or TSC2 mutant cells than the wild type cells. Two-week low dilution clonogenic assays in TSC1 or TSC2 mutant cells showed clone numbers were significantly reduced by rapamycin, RM-001, and RM-006, whereas in the wild type cells, Rapamycin and RM compounds had less reduction in clone numbers, indicating a differential sensitivity in TSC-null cells. Both RM-001 and RM-006 at ~1 nM showed near complete inhibition of p4E-BP1T37/46, which was not seen with rapamycin treatment, while inhibition of pS6S240/244 levels was similar for rapamycin and RM compounds. Further, the RM compounds had prolonged anti-proliferative activity in TSC-null cells, as shown by washout studies. Rapamycin, MLN0128 and both RM compounds markedly reduced kidney tumor burden in Tsc2+/- A/J mice after four weeks of treatment. Tumor regrowth was assessed two months after treatment cessation, tumor burden was significantly reduced in the RM-006 group as compared to the rapamycin and MLN0128 groups. Finally, TUNEL staining showed RM-006 treatment led to a greater induction of apoptosis in kidney tumor cells relative to rapamycin and MLN0128 after a single dose of each compound in Tsc2+/- A/J mice. This is the first time that clear evidence for cell death after mTORC1 inhibition has been seen in TSC rodent models. In summary, RM-001 and RM-006 demonstrate improved in vitro and in vivo inhibition of mTORC1 in comparison to rapamycin, and induced more cell death in TSC2 null tumors in vivo, indicating the potential of bi-steric mTORC1-selective inhibitors as a novel therapeutic strategy to treat tumors with mTORC1 dysregulation. Citation Format: Heng Du, Yu Chi Yang, Mallika Singh, Heng-jia Liu, David Kwiatkowski. Bi-steric mTORC1-selective inhibitors demonstrate improved potency and efficacy in tumors with mTORC1 hyperactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1026.

Marine-Derived Natural Products as ATP-Competitive mTOR Kinase Inhibitors for Cancer Therapeutics.

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase portraying a quintessential role in cellular proliferation and survival. Aberrations in the mTOR signaling pathway have been reported in numerous cancers including thyroid, lung, gastric and ovarian cancer, thus making it a therapeutic target. To attain this objective, an in silico investigation was designed, employing a pharmacophore modeling approach. A structure-based pharmacophore (SBP) model exploiting the key features of a selective mTOR inhibitor, Torkinib directed at the ATP-binding pocket was generated. A Marine Natural Products (MNP) library was screened using SBP model as a query. The retrieved compounds after consequent drug-likeness filtration were subjected to molecular docking with mTOR, thus revealing four MNPs with better scores than Torkinib. Successive refinement via molecular dynamics simulations demonstrated that the hits formed crucial interactions with key residues of the pocket. Furthermore, the four identified hits exhibited good binding free energy scores through MM-PBSA calculations and the subsequent in silico toxicity assessments displayed three hits deemed essentially non-carcinogenic and non-mutagenic. The hits presented in this investigation could act as potent ATP-competitive mTOR inhibitors, representing a platform for the future discovery of drugs from marine natural origin.

Molecular Modelling, Synthesis and Biological Evaluation of Novel Benzimidazole Derivatives for the Treatment of Breast Cancer

Background: The treatment of cancer requires scientific advancement. ATP-competitive mTOR inhibitors have been studied as potential antitumor agents. Objective: A series of substituted benzimidazole compounds were designed, synthesized and characterized via introducing 2-chloroquinolin into 2nd position and most title compounds exhibited enhanced anticancer activities. Methods: To study the anticancer mechanism, VIa-VIh was successfully docked by iGEMDOCK 2.0 which gives good affinity towards m-TOR/PI3K dual inhibitors. The anti-proliferative activities of these compounds were evaluated on MCF-7 and A549 cell line for Breast and lung cancer, respectively. Results: 2-(2-chloroquinolin-3-yl)-1H-benzoimidazol-1-yl)(phenyl)methanone (VIa) exhibited significant anti-proliferative activity, especially against breast cancer (IC50 197 μM) for MCF7 cell line and (2-(2- chloroquinolin-3-yl)-1H-benzo[d]imidazol-1-yl)(4-nitrophenyl)methanone (VIc) was significantly active against lung cancer (IC50 89 μM) for A579 cell line. Conclusion: VIa gives more activity on breast cancer and it gives IC50 197 μM for MCF7 cell line and (2- (2-chloroquinolin-3-yl)-1H-benzo[d]imidazol-1-yl) (4-nitrophenyl) methanone (VIc) lung cancer IC50 89 μM for A579 cell line.

De Novo Transcriptome Profiling of Brain Tissue from the Annual Killifish Nothobranchius guentheri.

Nothobranchius is a genus of small annual killifish found in Africa. Due to the relatively short lifespan, as well as easy breeding and care, Nothobranchius fish are becoming widely used as a vertebrate model system. Studying the genome and transcriptome of these fish is essential for advancing the field. In this study, we performed de novo transcriptome assembly of brain tissues from Nothobranchius guentheri using Trinity. Annotation of 104,271 potential genes (with transcripts longer than 500 bp) was carried out; for 24,967 genes (53,654 transcripts), in which at least one GO annotation was derived. We also analyzed the effect of a long-term food supplement with Torin 2, second-generation ATP-competitive inhibitor of mTOR, on the gene expression changes in brain tissue of adult N. guentheri. Overall, 1491 genes in females and 249 genes in males were differently expressed under Torin 2-supplemented diet. According to the Gene Set Enrichment Analysis (GSEA), the majority of identified genes were predominantly involved in the regulation of metabolic process, dendritic spine maintenance, circadian rhythms, retrotransposition, and immune response. Thus, we have provided the first transcriptome assembly and assessed the differential gene expression in response to exposure to Torin 2, which allow a better understanding of molecular changes in the brain tissues of adult fish in the mTOR pathway inhibition.

A systems pharmacology approach to identify the autophagy-inducing effects of Traditional Persian medicinal plants

Aging is correlated with several complex diseases, including type 2 diabetes, neurodegeneration diseases, and cancer. Identifying the nature of this correlation and treatment of age-related diseases has been a major subject of both modern and traditional medicine. Traditional Persian Medicine (TPM) embodies many prescriptions for the treatment of ARDs. Given that autophagy plays a critical role in antiaging processes, the present study aimed to examine whether the documented effect of plants used in TPM might be relevant to the induction of autophagy? To this end, the TPM-based medicinal herbs used in the treatment of the ARDs were identified from modern and traditional references. The known phytochemicals of these plants were then examined against literature for evidence of having autophagy inducing effects. As a result, several plants were identified to have multiple active ingredients, which indeed regulate the autophagy or its upstream pathways. In addition, gene set enrichment analysis of the identified targets confirmed the collective contribution of the identified targets in autophagy regulating processes. Also, the protein–protein interaction (PPI) network of the targets was reconstructed. Network centrality analysis of the PPI network identified mTOR as the key network hub. Given the well-documented role of mTOR in inhibiting autophagy, our results hence support the hypothesis that the antiaging mechanism of TPM-based medicines might involve autophagy induction. Chemoinformatics study of the phytochemicals using docking and molecular dynamics simulation identified, among other compounds, the cyclo-trijuglone of Juglans regia L. as a potential ATP-competitive inhibitor of mTOR. Our results hence, provide a basis for the study of TPM-based prescriptions using modern tools in the quest for developing synergistic therapies for ARDs.

Harmine and Piperlongumine Revert TRIB2-Mediated Drug Resistance.

Simple SummaryPoor survival and treatment failure of patients with cancer are mainly due to resistance to therapy. Tribbles homologue 2 (TRIB2) has recently been identified as a protein that promotes resistance to several anti-cancer drugs. In this study, RNA sequencing and bioinformatics analysis were used with the aim of characterizing the impact of TRIB2 on the expression of genes and developing pharmacological strategies to revert these TRIB2-mediated changes, thereby overcoming therapy resistance. We show that two naturally occurring alkaloids, harmine and piperlongumine, inverse the gene expression profile produced by TRIB2 and sensitize cancer cells to anti-cancer drugs. Our data suggest that harmine and piperlongumine or similar compounds might have the potential to overcome TRIB2-mediated therapy resistance in cancer patients.Therapy resistance is responsible for most relapses in patients with cancer and is the major challenge to improving the clinical outcome. The pseudokinase Tribbles homologue 2 (TRIB2) has been characterized as an important driver of resistance to several anti-cancer drugs, including the dual ATP-competitive PI3K and mTOR inhibitor dactolisib (BEZ235). TRIB2 promotes AKT activity, leading to the inactivation of FOXO transcription factors, which are known to mediate the cell response to antitumor drugs. To characterize the downstream events of TRIB2 activity, we analyzed the gene expression profiles of isogenic cell lines with different TRIB2 statuses by RNA sequencing. Using a connectivity map-based computational approach, we identified drug-induced gene-expression profiles that invert the TRIB2-associated expression profile. In particular, the natural alkaloids harmine and piperlongumine not only produced inverse gene expression profiles but also synergistically increased BEZ235-induced cell toxicity. Importantly, both agents promote FOXO nuclear translocation without interfering with the nuclear export machinery and induce the transcription of FOXO target genes. Our results highlight the great potential of this approach for drug repurposing and suggest that harmine and piperlongumine or similar compounds might be useful in the clinic to overcome TRIB2-mediated therapy resistance in cancer patients.

Drug repurposing of bladder cancer driven by patients’ proteomic signatures

Molecular signatures may present sources of druggable targets not adequately explored yet. The objective of this study was the identification of known de-risked potential therapeutic compounds (drug repurposing) for the treatment of high-risk NMIBC and MIBC, based on high resolution proteomics profiles. The next generation Connectivity Map (CMap) was employed for drug repurposing using as input proteomic signatures from patients with MIBC, and NMIBC of low (NPS3) and high aggressive molecular profile (NPS1) from our recently published datasets (Stroggilos et al. Int J Cancer.2020;146(1):281). The retrieved candidate drugs were ranked according to the disease–drug connectivity score. The impact of selected drugs was investigated in vitro in a panel of multi-origin BC cell lines including benign (HBLAK), non–muscle invasive (BFTC-905, SW1710) and invasive (T24, T24M, VmCub1, 253J, HT-1376) cells. The impact on cell proliferation (MTS), colony formation (matrigel), and apoptosis (Annexin V) was assessed. Among the top ranked compounds reversing the molecular signature of NMIBC from high to low risk subtype were mTOR inhibitors, tubulin inhibitors, caspase activators and RAF inhibitors. WYE-354, an ATP-competitive inhibitor of mTOR, had the highest connectivity score. WYE-354 was also ranked among the top candidate drugs when using as input proteins with consistent changes at the mRNA level in NMIBC progressors versus non-progressors from earlier published studies or proteomics changes in MIBC versus NMIBC. In vitro administration of WYE-354 resulted in a significant reduction in the proliferation rate of all the tested BC cell lines in a concentration-dependent manner and also impaired colony growth by significantly decreasing the size of the colonies. However, no significant effect on BC cell apoptosis could be observed. In this study, we describe a promising pipeline for identifying repurposed drugs of potential therapeutic impact for aggressive NMIBC, based on patients’ proteomic signatures. Deciphering the molecular mechanism of the impact of WYE-354 based on the molecular characteristics of the tested cell lines is ongoing. Evaluation of further compounds or combinations predicted by the CMap analysis is also planned.

A Novel Pipeline for Drug Repurposing for Bladder Cancer Based on Patients' Omics Signatures.

Simple SummaryCurrent therapies for bladder cancer present limitations as, regularly, not all patients benefit from treatment. Drug repurposing refers to the use of available drugs in new disease contexts. In this study, we identified drugs of potential to be repurposed, e.g., used in the treatment of aggressive bladder cancer. This was achieved by using earlier described molecular profiles (mRNAs, proteins) of tissue of patients with bladder cancer, matched to molecular profiles induced by drugs via mathematical tools. Through this process, multiple drugs potentially treating aggressive bladder cancer were predicted. Of these, an inhibitor of the mTOR molecular pathway was further investigated and found to significantly delay the growth of many bladder cancer cell lines. Collectively, this study provides a robust pipeline for drug repurposing based on tissue molecular profiles and highlights drug candidates meriting further experimental investigation in the treatment of aggressive bladder cancer.Multi-omics signatures of patients with bladder cancer (BC) can guide the identification of known de-risked therapeutic compounds through drug repurposing, an approach not extensively explored yet. In this study, we target drug repurposing in the context of BC, driven by tissue omics signatures. To identify compounds that can reverse aggressive high-risk Non-Muscle Invasive BC (NMIBC) to less aggressive low-risk molecular subtypes, the next generation Connectivity Map (CMap) was employed using as input previously published proteomics and transcriptomics respective signatures. Among the identified compounds, the ATP-competitive inhibitor of mTOR, WYE-354, showed a consistently very high score for reversing the aggressive BC molecular signatures. WYE-354 impact was assessed in a panel of eight multi-origin BC cell lines and included impaired colony growth and proliferation rate without any impact on apoptosis. Overall, with this study we introduce a promising pipeline for the repurposing of drugs for BC treatment, based on patients’ omics signatures.

GDC-0349 inhibits non-small cell lung cancer cell growth

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related human mortality with a clear need for new therapeutic intervention. GDC-0349 is a potent and selective ATP-competitive mTOR inhibitor. In A549 cells and primary human NSCLC cells, GDC-0349 inhibited cell growth, proliferation, cell cycle progression, migration and invasion, while inducing significant apoptosis activation. Although GDC-0349 blocked Akt-mTORC1/2 activation in NSCLC cells, it also exerted cytotoxicity in Akt1-knockout A549 cells. Furthermore, restoring Akt-mTOR activation by a constitutively-active Akt1 only partially attenuated GDC-0349-induced A549 cell apoptosis, indicating the existence of Akt-mTOR-independent mechanisms. In NSCLC cells GDC-0349 induced sphingosine kinase 1 (SphK1) inhibition, ceramide accumulation, JNK activation and oxidative injury. Conversely, N-acetylcysteine, the JNK inhibitor and sphingosine 1-phosphate alleviated GDC-0349-induced NSCLC cell apoptosis. In vivo, daily oral administration of GDC-0349 potently inhibited NSCLC xenograft growth in mice. Akt-mTOR in-activation, SphK1 inhibition, JNK activation and oxidative stress were detected in NSCLC xenograft tissues with GDC-0349 administration. In summary, GDC-0349 inhibits NSCLC cell growth via Akt-mTOR-dependent and Akt-mTOR-independent mechanisms.

Research progress of mTOR inhibitors.

Mammalian target of rapamycin (mTOR) is a highly conserved Serine/Threonine (Ser/Thr) protein kinase, which belongs to phosphatidylinositol-3-kinase-related kinase (PIKK) protein family. mTOR exists as two types of protein complex: mTORC1 and mTORC2, which act as central controller regulating processes of cell metabolism, growth, proliferation, survival and autophagy. The mTOR inhibitors block mTOR signaling pathway, producing anti-inflammatory, anti-proliferative, autophagy and apoptosis induction effects, thus mTOR inhibitors are mainly used in cancer therapy. At present, mTOR inhibitors are divided into four categories: Antibiotic allosteric mTOR inhibitors (first generation), ATP-competitive mTOR inhibitors (second generation), mTOR/PI3K dual inhibitors (second generation) and other new mTOR inhibitors (third generation). In this article, these four categories of mTOR inhibitors and their structures, properties and some clinical researches will be introduced. Among them, we focus on the structure of mTOR inhibitors and try to analyze the structure-activity relationship. mTOR inhibitors are classified according to their chemical structure and their contents are introduced systematically. Moreover, some natural products that have direct or indirect mTOR inhibitory activities are introduced together. In this article, we analyzed the target, binding mode and structure-activity relationship of each generation of mTOR inhibitors and proposed two hypothetic scaffolds (the inverted-Y-shape scaffold and the C-shape scaffold) for the second generation of mTOR inhibitors. These findings may provide some help or reference for drug designing, drug modification or the future development of mTOR inhibitor.

The Synergistic Effect of an ATP-Competitive Inhibitor of mTOR and Metformin on Pancreatic Tumor Growth.

ABSTRACTBackgroundThe mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensing pathway and a key regulator of amino acid and glucose metabolism. Dysregulation of the mTOR pathways is implicated in the pathogenesis of metabolic syndrome, obesity, type 2 diabetes, and pancreatic cancer.ObjectivesWe investigated the impact of inhibition of mTORC1/mTORC2 and synergism with metformin on pancreatic tumor growth and metabolomics.MethodsCell lines derived from pancreatic tumors of the KPC (KrasG12D/+; p53R172H/+; Pdx1-Cre) transgenic mice model were implanted into the pancreas of C57BL/6 albino mice (n = 10/group). Two weeks later, the mice were injected intraperitoneally with daily doses of 1) Torin 2 (mTORC1/mTORC2 inhibitor) at a high concentration (TH), 2) Torin 2 at a low concentration (TL), 3) metformin at a low concentration (ML), 4) a combination of Torin 2 and metformin at low concentrations (TLML), or 5) DMSO vehicle (control) for 12 d. Tissues and blood samples were collected for targeted xenometabolomics analysis, drug concentration, and cell signaling.ResultsMetabolomic analysis of the control and treated plasma samples showed differential metabolite profiles. Phenylalanine was significantly elevated in the TLML group compared with the control (+426%, P = 0.0004), whereas uracil was significantly lower (–38%, P = 0.009). The combination treatment reduced tumor growth in the orthotopic mouse model. TLML significantly decreased pancreatic tumor volume (498 ± 104 mm3; 37%; P < 0.0004) compared with control (1326 ± 134 mm3; 100%), ML (853 ± 67 mm3; 64%), TL (745 ± 167 mm3; 54%), and TH (665 ± 182 mm3; 50%) (ANOVA and post hoc tests). TLML significantly decreased tumor weights (0.66 ± 0.08 g; 52%) compared with the control (1.28 ± 0.19 g; 100%) (P < 0.002).ConclusionsThe combination of mTOR dual inhibition by Torin 2 and metformin is associated with an altered metabolomic profile and a significant reduction in pancreatic tumor burden compared with single-agent therapy, and it is better tolerated.